Phagocytic NADPH oxidase (Phox) is critically important for generating reactive oxygen species with microbicidal activity. Recently, nonphagocytic NADPH oxidase (Nox) isoforms have been identified which generate reactive oxygen species that function in signal transduction. Both isoforms are activated by lipid second messengers such as phosphatidylinositols and phosphatidic acid which recruit cytosolic components of the oxidase to the membrane. The goal of this project is to determine the structural basis for recognition of these activating lipids by the peripheral membrane proteins p47phox, p40phox, and the p47phox homolog NOXO1. This proposal teams a structural biologist with an interest in immune cell signaling with a coinvestigator with an extensive track record of functional analysis of Phox regulation by bioactive lipids. Aim 1 is to determine the structural basis of lipid recognition by the PX domains of p47phox, p40phox, and NOXO1. While crystal structures indicate the basis for soluble lipid head group interactions, information regarding the interfaces required for interaction with a lipid surface is lacking. Our preliminary data demonstrate that isotropic, phospholipid "bicelles" are a suitable membrane mimetic that will allow high-resolution, solution-state NMR structure determination of phospholipid bilayer-bound protein. Aim 2 tests the hypothesis that intramolecular associations within p47phox and NOXO1 regulate lipid binding by these proteins. Preliminary data show that phosphatidic acid binds to the polybasic region of p47phox, which is lacking in NOXO1. In addition, binding of phosphoinositides to the full-length protein is less effective than binding to the isolated PX domain. Both the polybasic region and the PX domain appear to interact with the SH3 domains, raising the possibility that binding of lipids and/or other modifications (e.g. phosphorylation of the polybasic region) alters the conformation of the proteins to allow more effective interaction with lipids and proteins in the membrane. These experiments will provide key insights into how oxidase activity is regulated and the differences in regulation and activation of phagocytic vs. nonphagocytic forms of the oxidase.